BIOCHEMICAL APPROACHES TO AGING:  Norton Rothstein

CONFOUNDING VARIABLES: MAMMALIAN SPECIES

1.  Short-lived strains might not manifest age related conditions.

2.  Strain differences produce possibly different aging characteristics.

3.  Diet and other conditions can cause variation in life span even of the same strain of rats.

4.  Rats should be raised pathogen free (barrier-reared).   Such animals tend to be longer lived,

thus implicating infectious agents in shortening life span.

5.  Some strains of rats with age tend to be lean while others become obese.

6. Environmental factors such as crowding affect longevity.

CONFOUNDING VARIABLES:  CELLS IN CULTURE

1.  While cells may be derived from almost any species and any tissue type, and may be stored

for long periods in liquid nitrogen; there is an unproven relationship between cells in culture and

aging in vivo.

2.  The number of population doublings drops significantly (40 to 28 using skin for 15 versus

75-year olds, though less if diabetics are removed) its relevance to aging is not clear, for if an

aged subject undergoes even 70% of their original potential for doubling, there does not appear

to be a serious danger of running out of cells.

3.  Because SV-40 virus can transform even late-passage (where there is a stead decline in the

number of cells doubling) into an "immortal" line, the relation between cell aging and aging is

even further obscured.

4.  Cell loose specialize cells and are repopulated by stem cells regardless of the tissue of origin.  This makes

it hard to correlate such in vitro cells, with in vivo cells.

5.  As cells divide and redivide there occurs a selection in which specialized cells tend to die out, and the

culture adapts to the medium.

6. There can be significant variation in cells from biopsy to biopsy, even in the same subject.

7.  Cells are being stressed in vitro to highly unnatural conditions thus possibly confounding a relationship

to aging in vivo.

8.  It has been repeatedly shown that the life-span of cells in vitro are determined by the cumulative number

of population doublings and not the calendar time in culture-­counter to in vivo.

9. Cell cultures, even from cloning experiments are heter­ogeneous; i.e., some are dividing rapidly, some

slowly, and some not dividing.

10.  No one has yet succeeded in making non-dividing cells divide normally.

11.  It has been found in human skin fibroblast that the cells centrally located existed at a much lower

generation level than those at the circumference, which divided more frequently.  This might help explain

the differences in pro-life active capacity found in sub cultured ceils.

12.  Serum used in culture medium, trypsin used to free cells from culture vessels and other conditions might

affect culture growth.

13.   Cortisone and hydrocortisone extended life span of human cells in culture.  Early passage cells where most

responsive, yielding a 30-40% extension.  It was inhibitory, or less effective on older cells, presumable because such

cells process decreased (about 40%) sites.

14.  Only 2%of cells from old donors could produce colonies of 256 cells or more, whereas

the majority of cells from young donors could do so.

a.  population doublings

b.  ability to maintain population, 10  cells/cm

c.  two weeks after plating at low cell densities

14.  In vitro cells do not as in vivo cells do have increase in content and size of tRNA.

15.  Similarly for residual bodies there is no increase in aged donors.

16. Unfortunately when preparing in vivo cells they are generally passage a few times before

use, thus making the difference between the two obscure.

17. Confluence is used to determine Phase III.  A cell population is harvested when a monolayer

covers a vessel (confluency), after about 45 doublings (this period termed phase II) the cells will

not reach confluence, their number decreasing with each passage, Phase III. 

18.  A number of biochemical changes have been observed, much more needs to be done.  No distinction was made as to whether the change occur in Phase II or Phase III cells, and some studies do not differentiate age of donor.

19.  The relationship between life span and potential for population doublings has not yielded

any conclusive results from the few studies that have been done.

20.  Experiments attempting to show weither the control of the potential for doubling resides in

the cytoplasm or nuclei have proven inconclusive.  For example, such factor for the WI-38 cells

appears either to lie in either the cytoplasm or in act­ivators of the nucleus.   These where

determined in transformed cell experiment in which parts of Phase II and Phase III cells where

fused.

21.  With tissue transplants, the age of the donor does not seem to be important, but rather the age

of the recipient: none did well with old recipients. Grafts have survived nearly 2 times the life span

of the animals, and others have obtained in longer lasting results.  Affects of the procedure

infusions of blood vessels and connective tissue confound conclusions.

22. Red blood cells, particularly human erythrocytes, since they cannot synthesize new

components for they are without nucleus, must survive on its original complement of structural and function elements.  The subsequent deterioration of enzymes and membranes during its life span

of 120-130 days can be regarded as a model for deteriorative processes in aging.  The lack of

repair

and spec deteriorative mechanisms are present confound conclusions.

23. It is difficult to resolve if related changes observed in experiments are reflective of their

significance in humans, plus the extremely subtle nature of the changes make progress difficult.

LIPIDS:

1.  The generation of free radicals occurs in biological systems and can be brought about by

ionizing radiation, UV radiation or by certain enzymatic and nonenzymatic reactions.

2.  Biological systems are protected from these effects b; a number of radical scavenging

molecules and by enzyme systems designed to remove superoxide radicals or peroxides

which result from free radical reactions.

3.  Skin spots are one sign of the incomplete protection these systems afford.

4.  Large doses of dietary antioxidants can function to reduce the amount of free radicals,

although extension of life span is not affected — at least in mammals.

5.  Much more basic information remains to be obtained before on can begin to put even a tentative picture as to what effect lipid metabolism has on aging and vice versa.

6.  In addition to free radicals, superoxide radicals are generated by a number of processes both

biological and chemical, including auto oxidation of hydroquinone, leukoflavins,

catecholamines, thiols, reduced dyes, tetra-hydropteridines, and ferredoxins, and as for

enzymes they include xanthine oxidase, aldehyde oxidase, NADH and NADP oxidase, certain

flavoprotein dehydrogenases, etc.

7.  Lipid peroxidation results in the formation of

malonaldehyde and thence age pigment.

8.   Free radicals have the potential to initiate in addition to malonaldehyde formation, addition,

scissions cross-linking, and aromatic hydroxylations, and these reactions could result in

mutagenic and carcinogenic effects, transcription errors, DNA damage, and protein inactivation. 

Though there is little unequivocal evidence to show that these reactions are major events in the

aging process, the idea of a small but steady burden on the tissues cannot be excluded.

9.  Hydroxyl radical is considered to be responsible for 90% of the damage to DNA caused by

ionizing radiation and that H₂0_? causes strand breakage.

10.  Age pigments, generally known as LIPOFUSCIN are believed to be derived from subcellular organelles as a result of cross-linking of membranes--the cross-linking is thought to be derived

from peroxidation of lipids.  Since the cross-linked products are not readily susceptible to the

action of proteolytic enzymes, they accumulate with time.

11. The amount accumulated can in terms of intracellular volume be quite significant, from

6 to 50% in very old animals depending on tissue.

ANTIOXIDANTS

1. Feeding mice a variety of antioxidants has caused an improvement in mean life span, which

appears to be species-related rather than general.

2.  What role antioxidants play in lengthening life is yet to be determined.

3.  Vitamin E reduce the lipid peroxidation, and unsaturated fatty acids increased it (and

therefore pigment: format ion).

4.  Studies with vitamin E suggest that age pigment accumulation is neither life threatening or

even seriously debilitating.

5.  Vitamin C has no effect on brain pigment formation.

6.  Little work on the direct effects of vitamin C on aging has been done.

7.  Centrophenoxine has been shown to reduce age pigments.

8.  Age pigment represents a mechanical accumulation of indigestible material, a result,

(and possible burden) rather than a cause of aging.

CHOLESTROL

1.  For men serum level increased from 177mg/dl to 248, from 17 to 55 years, then leveled

off; rapid rise occurring between 20 & 39.

2.  For women A similar pattern with a substantial jump in concentration between

49 and 59, with a higher level (285 vs. 259) in the 60 to 69 year age group,

3. Results with rats varied greatly for cholesterol: increasingly dependent on strain tested,

and even the very laboratory.

4.  Food restriction sharply reduced and delayed the degree o of increase.

MEMBRANES

1.  Though there are age related changes in membranes (mitochondrial function, enzyme

activity, lipid composition of mitochondrial and microsomal membranes) no one knows

if they initiate and develop the process of aging or are simply a result of the process.

2. If age related change occurs to membrane, their nature varies with type of membrane

involved.

3.  Effects of diet can bring about as great a change in membrane composition as aging.

4.  Age-related changes might change the susceptibility of membranes to fragmentation

and this would yield dissimilar preparations for analysis, or even different lipid adherence.

5.  Membrane turnover does not appear to change with age.

6.               There are disturbing contradictions and lack of con­sistency in results, from

7.               author to author.

             MITOCHONDRIA, MICROSOMES, & LYSOSOMES

1.  Certain enzyme levels in microsomes show age-related changes, but it is not known

if they result from membrane changes, altered enzymes, or a reduction in the number of enzyme

molecules.

2.  Results are influenced by differences in sex, strain, and maintenance conditions, and tissue

examined.

3.  Finally there is no direct link that has been estab­lished between enzyme changes and aging.

4. Enzyme changes could be secondary to changes related to gene expression, studies of

mitochondrial and microsomal metabolism in old organisms should point the invest­igation to

these primary facets of aging.

5.  For mitochondria there is a reduction in the number

and volume density with age.  There is also a fraction of mitochondria with increased fragility in old preparations.